RATIONAL DRUG DESIGN
TEACHER NOTES
There are three separate tasks here that students can work on. Each task should be completed within approximately 40 minutes with 10 minutes using the instructional Power Point to give students background on the particular protein of choice. The Power Point presentation also covers aspects of protein structure and function. If this has already been covered in class you can skip ahead to the slide covering the protein you are interested in.
Students can explore drugs designed to target the activity of the following proteins:
· the enzyme amylase – developing a diet pill
· the flu enzyme Neuraminidase – developing Relenza, an antiviral drug for the flu
· The Acetylcholine receptor channel – blocking sensory nerve impulses
Designing a Diet Pill
1. Name some foods that have a high content of starch. Rice, potato, cereals, etc
2. Choose from the list of words below to fill in the blanks for the following statements:
The enzyme amylase breaks down starch into disaccharide molecules called MALTOSE
The enzyme maltase breaks down maltose into the monosaccharide GLUCOSE
Amylase is produced by the salivary glands and the PANCREAS
The monosaccharide glucose is required by our bodies to provide ENERGY
Open the Cn3D file named ‘amylase’
3. How many alpha helices are there in this polypeptide? 8
4. How many beta sheets are there in this polypeptide? Tell them to use the sequence alignment viewer to count these. Count the number of brown amino acid sequence blocks = 28
5. What parts of the enzyme appear to be making up:
(a) the entrance to the active site? These are the random coils or random loops that are cradling the drug
(b) the active site? The actual active site is made up of a ring of beta sheets (the cofactor chloride ion is also found in the active site)
6. Charged ions are often required to assist an enzyme to do its job. These ions are cofactors. Which cofactor seems to be involved in the functioning of amylase (is in the active site)? Cl-
7. Sugar units often form a ring shape. How many sugar units are there in the drug molecule (the larger molecule seen here)? 6
8. What organism was this enzyme found in, and what part of the organism? Human pancreas
9. The large carbohydrate in this molecule is an inhibitor molecule. It stops the enzyme from breaking down starch. Looking at the location of the inhibitor, how might it be exerting its effect? It is blocking the entrance to the active site so the substrate starch can no longer get into the active site (a little like rolling a rock in front of a tunnel)
10. Explain how the drug shown interacting with amylase would help someone to lose weight. When we eat foods high in starch, such as bread, amylase breaks down the starch into maltose. This is in turn broken down by the enzyme maltase into glucose. Glucose is used to provide cells with energy in respiration. If we have more glucose than the cells need, it is stored as glycogen in the liver and as fat in adipose tissue.
If you stop amylase from working, then starch will pass through the body without being digested. As a result there will be less glucose available so less will be stored as fat.
11. Do you think the drug being designed to inhibit the action of amylase would block the active site of this enzyme reversibly or irreversibly? Explain your answer. Would be best if it is reversible so that some starch is broken down. We don’t want to permanently block amylase or we could end up not having enough glucose for cell metabolism.
Saving Lives: Designing a Flu Drug
It is really important that you ensure your students have made the connection between the N protein on the virus coat and the N protein they view in Cn3D. Often they will think they are looking at the whole virus when they open up N in Cn3D.
Remind them that N acts like a pair of scissors, cutting the links between the virus and the host cell membrane.
If they move the molecule around so the drug is at the top, they can see that it looks like its being cradled in a cup-like indentation. The important part (the active site) or the blades of the scissors is shown here being blocked by the drug.
If they think of the active site like the scissor blades we can use an analogy. If you put a rock between scissor blades, can they still cut? No. If we place a rock (the drug) in the active site of N, can it still cut? No. So the virus cannot escape the host cell which means that it won’t spread.
What they need to think about is that if we want to block the active site, the drug we design must interact more strongly with one or more of the amino acids in the active site that the normal substrate does. In this way the drug will competitively bind with the active site rather than the substrate, sialic acid.
Open the Cn3D file named ‘Neuraminidase’
12. Complete the following statement by circling the correct response.
Neuraminidase protein is composed of random coils and alpha helices / beta sheets.
13. Locate a disulfide bond found in Neuraminidase. Double click on the two amino acids making up this bond. Find out the one letter code for the amino acids in this bond and then use the table below to find out the name of the amino acid. C = Cysteine
1- letter code / Name of amio acid / 3-letter code / 1- letter code / Name of amio acid / 3-letter codeG / Glycine / Gly / P / Proline / Pro
A / Alanine / Ala / V / Valine / Val
L / Leucine / Leu / I / Isoleucine / Ile
M / Methionine / Met / C / Cysteine / Cys
F / Phenylalanine / Phe / Y / Tyrosine / Tyr
W / Tryptophan / Trp / H / Histidine / His
K / Lysine / Lys / R / Arginine / Arg
Q / Glutamine / Gln / N / Asparagine / Asn
E / Glutamic Acid / Glu / D / Aspartic Acid / Asp
S / Serine / Ser / T / Threonine / Thr
14. How many sugar groups are bound to Neuraminidase (do not include the drug molecule in this)? 3
15. The normal substrate that N acts on is called sialic acid. It enters the active site of N and is ‘stressed’ so bonds break cutting the virus free of the host cell so it can go off to infect more host cells. The drug you can see on your screen is Relenza. Sialic acid and Relenza are shown below. Circle any differences you can see on the Relenza drug molecule. These differences make it bind to the active site of N more strongly.
16. Using the sequence alignment viewer, place your cursor over the amino acids that are now yellow and read their location in the protein chain. Record their location in the table below. When you have done this use the amino acid table on page 4 to find out the name of each amino acid.
Location and name of highlighted amino acids:
Name of amino acid / 1-letter code / Location / Name of amino acid / 1-letter code / Location1. Arginine / r / 37 / 5. Glutamic acid / e / 147
2. Aspartic acid / d / 70 / 6. Glutamic acid / e / 196
3. Arginine / r / 71 / 7. Arginine / r / 212
4. Arginine / r / 144 / 8. Arginine / r / 290
Check your answer:
You should have 5 arginines (r), two glutamic acids (e) and one aspartic acid (d).
There are also three non-charged amino acids in the active site that also bind with the drug. They are Tryptophan (w), Isoleucine (i) and Tyrosine (y). The uncharged molecules are grey.
17. What is the function of N for the flu virus?
N functions to cut the flu virus away from the host cell so the virus can spread to infect more cells
18. Explain how the drug shown interacting with N stops the flu virus from spreading and infecting new host cells. The drug blocks the active site so the flu virus gets stuck to the host cell and can’t move off to infect more of your cells.
19. Would you design a drug to inhibit the action of N reversibly or irreversibly? Explain your answer. Ideally the drug will irreversibly bind to the active site so N can never work. The virus is forever stuck to the host cell and the host cell covered in virus would be engulfed by a macrophage.
20. The region of N that cuts the flu virus away from receptors on the host cell surface is known as the “active site”. The genetic code of the influenza virus mutates rapidly to help influenza escape our immune system. However, the regions of the genome that encode the “active site” are highly conserved. Discuss specificity of the active site to describe why this might be? If the shape of the active site changes then the action of the protein will no longer work. In this case, the N active site would no longer bind with its substrate, sialic acid, so the virus would never escape host cells to infect new cells. This mutation would not be passed on to subsequence viruses as they would never make it to a new host cell to replicate.
Controlling Chronic Pain: Venoms for Drugs
Open the Cn3D file named ‘Ion channel and neurotransmitter’
21. How many acetylcholine molecules are found binding with this molecule (this chemical causes the ion channel to open)? 1
22. How many parts make up this ion channel? (look for the different colours) 5 (5 polypeptides are found making up the quaternary structure)
23. Rotate the molecule so you have an aerial view. Draw 2 quick sketches showing what it looks like from the side and from above. Indicate on each where transport of sodium ions would occur.
24. Indicate on each sketch above where the acetylcholine binding site is located.
25. Would you expect negatively charged ions to be attracted to or repelled away from this ring of negatively charged amino acids? Explain your answer. Should be repelled as like charges repel.
26. Outside the cell there is a soup of positive and negative ions and other chemicals. Explain how the structure of this ion channel would stop negative ions from moving through the pore and entering the cell. This ring of negatively charged amino acids will attract positive ions like sodium and calcium – the ones that set of a nerve impulse. The negatively charged ones will be repelled leaving the pore entrance free for positive ions to move through.
27. How many disulfide bridges does each of these alpha-conotoxins have? 1
28. Quickly sketch each protein from the side showing the effect that binding of alpha-conotoxin exerts (NB/ Alpha-conotoxin changes the shape of the receptor protein so the pore in the ion channel won’t open. Our structure only shows the receptor, not the pore going through the cell membrane). See the first diagrams after the heading ‘controlling chronic pain: venoms for drugs’
Killer a conotoxins: We now know that the killer drugs have a loop of 3 amino acids and then 5 amino acids separated by a disulfide bond (a3/5). These drugs target nicotinic Acethylcholine Receptors in muscles so the heart and diaphragm are affected adversely.
Therapeutic a conotoxins: Alpha conotoxins that have 4 amino acids and 7 amino acids separated by a disulfide bond (a 4/7), act on neuronal nicotinic Acetylchoine Receptors found in the sensory nerves. These drugs do not kill.
Look at the sequence for an a conotoxin shown below:
e c c n p a c g r h y s c x
Disulfide bonds form between two cysteine amino acids (c-c). We need to count the number of amino acids between these c’s.
In the example above we have 3 and then 5 = (a3/5 conotoxin). It’s a killer!
29. Write down the sequences of alpha-conotoxin A and alpha-conotoxin B below and determine the name you would use for each (i.e. a ?/? conotoxin)
Alpha-conotoxin A: xccnpacgpkyscx a3/5 conotoxin
Alpha-conotoxin B: gccslppcaannpdycx a4/7 conotoxin
30. Which of these two conotoxins would you choose to develop as a drug for blocking peripheral nerve pain? Explain. Alpha conotoxin B as it is not a killer but blocks sensory nerve pain.
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